Abstract

This paper is to emphasize the higher antitumor effect of IL-24, because it can make the complete elimination of the xenograft tumor. Another important point in this paper is to introduce a strategy for essentially complete eradication all the xenograft tumor which was named “Cancer Targeting Double Gene-Viro-Therapy or the Cancer Targeting Gene-Viro- Therapy with double gene (CTGVT-DG)”.

Keywords

Introduction

A novel gene mda-7 (melanoma differentiation-associated
gene 7), was identified by subtraction hybridization using a human
melanoma cell line (HO-1) [1-5]. Because of structure of mda-7 similar
to the interleukin 10 (IL-10) family of cytokines with chromosomal
localization and cytokine-like properties, mda-7 has been redesignated
as IL-24 [2-5]. The mda-7 cDNA encodes a protein of 206 amino acids
with a predicted size of 23.8 kDa [6].

Many studies have shown that enforced expression of IL-24
suppresses cell growth and induces apoptosis in a variety of tumor types
including melanomas, gliomas, and cancers of the breast, colon, lung,
cervix, pancreas, and prostate [7-15]. In contrast, these investigations
also demonstrated that elevated expression of mda-7/IL-24 in normal
mammary epithelial cells has no cytotoxic effects [16,17]. Studies
showed that mda-7/IL-24 induces growth suppression and apoptosis in
diverse cancer cells [5,14]. In addition to its direct antitumor activity,
mda-7/IL-24 also exerts antiangiogenic activity in vivo [18].

Because IL-24 is a novel and prospective gene for the therapy of
multiple cancers, understanding the mechanism by which this gene
induces apoptosis in cancer cells will be of immense value. Studies are
beginning to high light on the signaling cascades involved in mda-7/
IL-24 induction of apoptosis [19-22]. Analyses of signaling pathways have revealed Ad-IL-24 regulation of inducible nitric oxide synthase
(iNOS) and mitogen-activated protein kinase (MAPK) in melanoma
[23] and Jun kinase, h-catenin, phosphatidylinositol 3-kinase (PI3K),
and protein kinase R(PKR) in lung and breast tumor cells [24,25]. In
the following text, I will describe the excellent antitumor effect of IL-24
on prostate cancer or hapatoma and the complete elimination of the
xenograft tumor by the CTGVT-DG strategy.

Study on the Excellent Antitumor Effect of IL-24 on
Prostate Cancer

We have made two constructs to study their antitumor effect: The
Ad·DD3·E1A·WPRE·E1B (Δ55)·(PTEN) (Figure 1a) [26] the Ad·IL-
24·DD3·E1A (Figure 1b) [27]. In (Figures 1a and 1b), both the DD3,
a prostate cancer specific promoter, were used to replace the native
promoter in E1A of adenovirus and drive the OncoAd (oncolytic virus from adenovirus) to specific targeting to prostate cancer and used the
same CMV promoter to drive the two expression cassettes of PTEN
gene and IL-24 gene. Although, PTEN is a brood cancer suppressor
gene with rather prostate cancer tropism, but we added a WPRE to enhance its activity of mRNA more stable and deletion 55 KD in E1B to
make the adenovirus more specific targeting to cancer cells as shown in
(Figure 1a). All of these were lack in (Figure 1b) which only the IL-24
was use without any helpless (Figure 1b) [27]. However, the xenograft
prostate cancer can be completely eliminated by the only IL-24 gene
(Figure 2b) [27]. Which is better than (Figure 2a) [26], showing the
excellent antitumor effect of IL-24.

Figure 1a: Construction of Ad·DD3·D55-PTEN. Schematic diagram of
Ad·DD3·D55-PTEN. The endogenous promoter of E1A was replaced by the
DD3 promoter, and the E1B-55K gene was deleted. The PTEN expression
cassette was inserted into Ad.DD3.D55 to construct Ad·DD3·D55-PTEN. ITR,
inverted terminal repeat.

Figure 1b: Construction of Ad·DD3-E1A-IL-24. Schematic diagram of the
viruses. In Ad·DD3-E1A, the endogenous promoter of E1A was replaced
with the DD3 promoter. The expression cassette of IL-24 was inserted into
Ad.DD3-E1A to construct Ad·DD3-E1A-IL-24. Ad-IL-24 is a replicationdeficient
adenovirus with a deleted E1 region. ITR, inverted terminal repeats;
ψ, packaging signal.

Figure 2a: Ad·DD3·D55-PTEN inhibits the growth of prostate cancer cell
xenograft tumors. Antitumor effect of infection of CL1 xenograft tumors with
different adenoviruses. The tumor volume was measured and is presented as
the mean ± SD.

Figure 2b: Antitumoral efficacy of the viruses in nude mice. PBS or different
viruses were intratumorally administered to nude mice bearing DU145
xenograft tumors. The tumor volumes (mean ± SD, n=6) were measured with
caliper and estimated using the following formula: tumor volume (mm3)=length
× width2/2.

Study on the excellent antitumor effect of IL-24 on hepatoma

In liver cancer, a specific promoter AFP was replaced the native
E1A promoter of adenovirus to make more specific to live cancer and deleted and the 55 KD of E1B in adenovirus was deleted to make
it more targeting to cancer cell, then we can make two products
(Figure 3): 1. Ad·enAFP·E1A·E1B(Δ55)·(Trail) (AFP·D55-Trail) and
2. Ad·enAFP·E1A·E1B(Δ55)·(SOCS3) (AFP·SOCS3-Trail)as shown
in (Figure 3a) [28], the Trail is a gene from TNFα superfamily, the
TNFSF10, which has good antitumor effect, but much less toxicity comparing with TNFα itself, the SOCS3 is good and rather liver specific
tropism antitumor gene.

By the combine of constructs 1 and 2, good anti-hepatoma effect
was obtained as shown in (Figure 3a) [28]. However, the antitumor
effect of two gene, the SOCS3 plus Trail as shown in (Figure 3b) was
less than that of only one IL-24 gene construct as shown in (Figures 4a and 4b) [29], showing also the super antitumor effect of IL-24 gene.

Figure 3b: The antitumor effect of the combined treatment of AFP-D55-SOCS3 and AFP-D55-TRAIL in vivo. (a) Tumor volumes were recorded. Each point represents
the means ± SD tumor volume (n=8). (b) Kaplan-Meier survival curves of animals. The pair-wise log-rank test was used to analyze the survival rates of different
groups. (c, d) At the end of this experiment, tumors removed from the mice were documented as photograph and weighted (**p<0.01)

Figure 4a: Ad.enAFP-E1A-ΔE1B-IL-24. Schematic structure of the recombinant oncolytic adenovirus. All viruses were created using the backbone of wild-type Ad5
(Ad-Wt). As for Ad·enAFP-E1A-ΔE1B-IL-24, the native E1A promoter was replaced by the AFP promoter modified with the SV40 enhancer at its 50 flank, and both
E1B-19 kDa and E1B-55 kDa genes were deleted to construct Ad·enAFP-E1A-ΔE1B, which was further modified with the interleukin (IL)-24 expression cassette driven
by the murine cytomegalovirus promoter (mCMV) to form the gene-virus Ad·enAFP-E1A-ΔE1B-IL-24. ITR is the inverted terminal repeats.

Figure 4b: Potent antitumor efficacy of Ad·enAFP-E1A-ΔE1B-IL-24 in nude mice. Female BALB/c nude mice were subcutaneously inoculated with Huh-7 cells at the head
and neck region (5 ×106 cells per 100 ml). When tumors reached a size of ~ 90mm3, the animals were treated with an intratumoral injection of Ad·enAFP-E1A-DE1B-IL-24 or
PBS (injected virus with a daily dose (4 × 108 plaque-forming units (PFU)) for 5 consecutive days). Data are presented as the mean ± s.d. (n=6). AFP, α-fetoprotein.

Excellent antitumor effect of the CTGVT-DG strategy

Currently there are cancer gene therapy and cancer oncolytic
virotherapy two fields. We innovate a third field, by inserting an
antitumor effect into an oncolytic virus (OV-gene) [30,31] and named
it as Cancer Targeting Gene-Viro-Therapy, CTGVT. The antitumor
effect of CTGVT (OV-gene) was very much increased than its original
two therapy. It is because that the gene can be induced to highly
replication by its vector OV’s replication. Therefore, the antitumor
effect CTGVT (OV-gene) was much increased [32]. However, if we use
two gene in the CTGVT (OV-gene) system i.e. the OV-gene1 plus OV-gene2
or OV-gene1-gene2 and named as CTGVT-DG, by which all the
xenograft tumor can be completely eradicated.

Here we only give an example of our CTGVT-DG strategy, the
ZD55-Trail+ZD55-Smac or the ZD55-Trail-IETD-Smac as shown in
(Figure 5) [33]. Since the two genes we used may have compensative or
synergetic effect between them many other CTGVT-DG to complete
eradication of xenograft tumor has been obtained by us [34-38].
The antitumor effect of the CTGVT-DG is much higher than that
of the antitumor effect of PD-1 antibody or Amgen’s excellent drug
OncoHSV-GM-CSF (data which will be published later). That is a great
success.

Figure 5: Antitumor efficacy of ZD55-TRAIL-IETD-Smac in nude mice. Female BALB/c nude mice were subcutaneously inoculated with Bel-7404 cells (1 × 107).
When tumors reached a size of 80-100mm3, the animals were treated with an intratumoral injection of ZD55-TRAIL-IETD-Smac or other recombinant adenovirus
at the dose described in Materials and Methods. PBS was used as a control. (A) Tumor size was measured and tumor volume was monitored at various times after
treatment. Data are presented as means ± SD (n=8). (B) Kaplan-Meier survival curves of animals. The percentage of surviving mice was calculated by monitoring
the death of mice over a period of 73 days. A pair-wise log-rank test was used to analyze survival rates in the various groups. (C and D) Representative histological
changes and protein expression of TRAIL and Smac in xenografts on day 7 after treatment. Tumors were harvested and fixed in 4% paraformaldehyde, embedded in
paraffin, cut into 4μm sections, and then subjected to immunochemical staining, TUNEL assay, and H&E staining (original magnification, × 200).